1. Field of the Invention
This invention relates to a method for enhancing the anti-infective activity of muramyldipeptide derivatives using liposomes.
2. Related Disclosures
In the field of immunology, multiple injections of a vaccine or bacterin are frequently required to produce the immunological response in a subject sufficient to ward off an infection. During the development of the vaccine art, researchers discovered that substances could be added to the vaccine composition which would enhance the immunogenicity of the vaccine, resulting in an immune response superior to that achieved by administering the antigen alone. Compounds which enhance the antigenicity of an antigen are known as adjuvants. Among the most effective adjuvants developed early in the vaccine art is Freund's complete adjuvant, which is a suspension of killed, whole Mycobacterium tuberculosis emulsified in mineral oil. Also highly effective is Freund's incomplete adjuvant, which is a mineral oil emulsion without mycobacteria. These two adjuvants are used as laboratory standards, but are not used commercially because Freund's complete adjuvant contains the microorganism M. tuberculosis, and both the complete and incomplete forms contain mineral oil, which is known to produce granulomas and other toxic effects. Also, in the absence of the mycobacteria, the incomplete adjuvant does not always produce a satisfactorily high immunogenic enhancement.
The basic units responsible for enhancing the cellular antigenicity response are believed to be the sugar-containing peptides of the mycobacterial cell wall. A detailed study of the chemistry of mycobacterial cell wall resulted in the observation by F. Ellouz, et al., Biochem. Biophys. Res. Comm., 59, 1317 (1974) that the adjuvant activity could be directly attributed to the bacterial wall peptidoglycan derivatives. The smallest effective molecule was found to be an N-acetylmuramyldipeptide, specifically N-acetylmuramyl-L-alanyl-D-isoglutamine. See C. Messer, P. Sinay, and A. Adams, Biochem. Biophys. Res. Comm., 66, 1316 (1975). This compound is now commonly called muramyldipeptide or MDP.
Subsequently, a number of muramyldipeptide analogs and derivatives were prepared by various academic and industrial concerns. The majority of these derivatives have been demonstrated to have some degree of immunopotentiating activity. In addition, many of these muramyldipeptide derivatives are per se active in enhancing the host immunity against infectious organisms such as Klebsiella pneumonia, Escherichia coli, Candida albicans, Staphylococcus aureus and the like. The literature on MDP derivatives and their immunological activities is extensive.
It is known that the anti-infective activity of muramyldipeptide derivatives may be enhanced by encapsulating the MDP derivative in liposomes. Liposomes are microscopic vesicles, generally spherically shaped, formed from one or several concentric bilayers (lamellae) of lipid mmolecules having a lipophilic and hydrophilic moiety. Most frequently, liposomes are water insoluble phospholipids which form bilayer structures spontaneously in aqueous suspension. Regardless of the overall shape, the bilayers are organized as closed concentric lamellae, with an aqueous layer separating each lamella from its neighbor. The lamellae of water-soluble liposomes comprise at least one lipid bilayer, the molecules of this layer being oriented so that the hydrophilic functions are in contact with the aqueous phase. Since the liposome layers are separated from each other by an aqueous film, they have a wall-like structure which can be schematically represented, in sections, by molecular composite XY-YX, X representing the hydrophilic portion of the molecule and Y the lipophilic portion. Liposome vesicle size is highly variable and dependent on the composition and method of manufacture, but generally ranges from 25 to 30,000 nm in diameter with a film thickness in the bilayer of 3 to 10 nm.
In recent years, liposomes have attracted widespread interest. Researchers have examined aspects ranging from theoretical physical chemistry to projected applications, particularly in medicine.
The physical chemistry studies have focused on such properties as fluidity, permeability, and molecular organization. These studies are generally motivated by the importance of the lipid bilayer as a structural analog of natural membranes. Liposomes may be used to alter membrane phospholipid and cholesterol content and transfer water-soluble, normally impermeant molecules into cells.
In clinical pharmaceutical research, liposomes have been viewed as a capsule for the possible selective delivery of therapeutic agents such as insulin, enzymes, and anti-tumor drugs.
Liposome-encapsulated muramyldipeptide derivatives have been used in studies wherein noncytotoxic macrophages have been rendered tumoricidal by the interaction of macrophage-activating factor and a free or encapsulated MDP derivative in studies reported by S. Sone and I. J. Fidler, The Journal of Immunology, 125 (6), 2454-2460 (1980). See also Sone and Fidler, Cellular Immunology, 57, 42-50 (1981); and I. J. Fidler, et al., Proc. Natl. Acad. Sci. USA, 78, 1680-1684 (1981). The in vivo studies of Fidler, et al., reported in Proc. Natl. Acad. Sci. USA teach that empty multilamellar vesicles with free MDP derivatives do not activate tumoricidal activity in murine alveolar macrophages when the free MDP derivative was administered at the same level as that adequate to cause activation by liposome-encapsulated MDP derivatives. These investigators found that a dose of free MDP derivative 80 times greater than the liposome-encapsulated MDP derivative dose did not activate the tumoricidal activity of murine alveolar macrophages.
It has been shown that the dose of an MDP derivative required for efficacy in protecting mice from bacterial or yeast infections can be reduced significantly by encapsulating the derivative in liposomes. For example, to achieve protection against Candida albicans yeast infection in mice, the MDP derivative dose was reduced by approximately 15-fold by encapsulating the MDP derivative in multilamellar liposome vesicles. Efficacy is also related to the liposome composition. This increased efficacy was only achieved when the liposome-encapsulated MDP derivative was given by intravenous injection, which allows the liposomes to be targeted to the phagocytic cells of the reticuloendothelial system. (See E. B. Fraser-Smith, et al., ASM meeting, March 1982.)
It has now been determined that this same increased efficacy can be achieved without actually encapsulating the derivative in liposomes, and without other physical association of the derivative with liposomes (such as association with the lipid bilayers) but by simply co-administering the MDP derivative with liposomes. The derivative can either be mixed with the preformed liposomes prior to injection, or the derivative injection may be preceded or followed by injection of the liposomes. Such co-administration of liposomes and free MDP derivatives provides essentially the same anti-infective activity as that obtained with the same dose of liposome-encapsulated MDP derivative, whereas administration of MDP derivative alone is ineffective at a similar concentration and does not impart anti-infective activity on an equivalent basis until the dose is increased by at least an order of magnitude.
Thus, one advantage of the invention is that one may obtain an enhanced anti-infective activity without introducing the desired MDP derivative during the liposome preparation, as is required by methods in the art.